Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 1B, hepatitis C virus genome-replicating cells transfected with the same, and method for producing infectious hepatitis C virus particles

a technology of hepatitis c virus and nucleic acid, which is applied in the field of nucleic acid constructs, can solve the problems of inability to compare the replication mechanism or replication efficiency of hcv between different genotypes, the difficulty of revealing the relationship between the hcv genotype and the replication mechanism or replication efficiency of hcv, and the limited type of hcv particles that can be artificially prepared as hcv vaccine raw materials,

Inactive Publication Date: 2016-01-12
JAPAN AS REPRESENTED BY DIRECTOR GENERAL OF NAT INST OF INFECT IOUS DISEASES +2
View PDF8 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0080]The present invention can provide an HCV subgenomic replicon RNA of genotype 1b that can be amplified in cultured cells and an HCV fullgenomic replicon RNA that can produce infectious HCV particles, which can be used in screening for an anti-HCV drug that inhibits infection and replication of HCV of genotype 1b, research for revealing the replication mechanism of HCV, and also development of an HCV vaccine based on HCV particles.

Problems solved by technology

It is therefore difficult to reveal a relationship between the HCV genotype and the replication mechanism or replication efficiency of HCV.
In addition, the type of HCV particles that can be artificially prepared as an HCV vaccine raw material is currently limited to the genotype 2a.
In studies using subgenomic replicon RNAs or fullgenomic replicon RNAs derived from HCV of the same genotype, the replication mechanism or replication efficiency of HCV cannot be compared between different genotypes, and it is difficult to identify a factor necessary for replication, which can be a novel target candidate of an anti-HCV drag, or to screen for an anti-HCV drug capable of showing a medicinal effect independent of the replication mechanism or replication efficiency.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 1B, hepatitis C virus genome-replicating cells transfected with the same, and method for producing infectious hepatitis C virus particles
  • Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 1B, hepatitis C virus genome-replicating cells transfected with the same, and method for producing infectious hepatitis C virus particles
  • Nucleic acid construct comprising nucleic acid derived from genome of hepatitis C virus of genotype 1B, hepatitis C virus genome-replicating cells transfected with the same, and method for producing infectious hepatitis C virus particles

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of HCV Subgenomic Replicon RNA Expression Vector of Wild-type NC1 Strain

[0216]An HCV subgenomic replicon RNA expression vector, plasmid pSGR-NC1, was constructed using the non-structural region of a cloned DNA (full-length genomic clone DNA) corresponding to the full-length genomic RNA of the NC1 strain, which is a novel HCV of genotype 1b separated from an acute severe hepatitis C patient.

[0217]Specifically, RNA was extracted from patient's serum and purified with Isogen-LS (Nippon Gene Co., Ltd.), and cDNA was synthesized using a random hexamer primer. PCR primers were designed based on the conserved sequence (Con1 strain: GenBank Accession No. AJ238799) of a known HCV genome of genotype 1b. Five fragments of cDNA were amplified using the designed PCR primers and the synthesized cDNA as a template. The amplification product of the sequence at the 5′ end, which is difficult to obtain, was obtained by a 5′ RACE method. Each fragment was cloned into a cloning vector for ...

example 2

Production of HCV Subgenomic Replicon RNA of Wild-type NC1 Strain

[0224]The expression vector pSGR-NC1 constructed in Example 1 was cleaved with restriction enzyme XbaI. Subsequently. 20 U of Mung Bean Nuclease was added to 10 to 20 μg of the XbaI cleaved fragment (the total amount of the reaction solution: 50 μL), followed by incubation at 30° C. for 30 minutes. Mung Bean Nuclease is an enzyme that catalyzes a reaction of smoothing through selective decomposition of the single-stranded portion of a double-stranded DNA. In general, RNA transcription by an RNA polymerase directly using the XbaI cleaved fragment as a template DNA synthesizes a replicon RNA having extra four nucleotides CUAG, a part of the Xbal recognition sequence, on the 3′ end. In this Example, accordingly, the XbaI cleaved fragment was treated with Mung Bean Nuclease to remove the four nucleotides CTAG from the XbaI cleaved fragment.

[0225]Subsequently, proteins in the Mung Bean Nuclease treated solution containing t...

example 3

Establishment of HCV Subgenomic Replicon-replicatiug Cell Clone of the NC1 Strain

[0227]Total cellular RNA extracted from Huh7 cells by a common method was mixed with 1 μg of the HCV subgenomic replicon RNA of the wild-type NC1 strain produced in Example 2 and the total RNA amount was adjusted to 10 μg. Subsequently, the RNA mixture was transfected into Huh7 cells by electroporation. The electroporated Huh7 cells were seeded in a culture dish and were cultured for 16 to 24 hours, and G418 (neomycin) was then added to the culture dish. The culture was continued while replacing the culture solution twice a week.

[0228]After the culture for 21 days after the seeding, viable cells were stained with crystal violet. As a result colony formation of the cells transfected with the HCV subgenomic replicon RNA of the NC1 strain was confirmed. The colony formation indicates that the HCV subgenomic replicon RNA was replicated in the cells (FIG. 2).

[0229]The colonies of viable cells were further cl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
nucleic acidaaaaaaaaaa
full-length genome nucleotide sequencesaaaaaaaaaa
structureaaaaaaaaaa
Login to View More

Abstract

A subgenomic replicon RNA (nucleic acid) having an excellent autonomously replicating ability and a fullgenomic replicon RNA (nucleic acid) having an excellent autonomously replicating ability and infectious HCV particle-producing ability, each derived from a novel HCV of genotype 1b, are provided. Particularly, a subgenomic replicon RNA (nucleic acid) and a fullgenomic replicon RNA (nucleic acid), each derived from an HCV genome of the NC1 strain which is a novel HCV genotype 1b isolated from a patient with acute severe hepatitis C, are provided.

Description

TECHNICAL FIELD[0001]The present invention relates to a nucleic acid construct containing a hepatitis C virus genome of genotype 1b, hepatitis C virus genome-replicating cells transfected with the nucleic acid construct, and a method of producing infectious hepatitis C virus particles.BACKGROUND ART[0002]In basic research for hepatitis C virus (hereinafter, also referred to as HCV) and development of anti-HCV drugs, an experimental system that can efficiently amplify HCV is essential. Specifically, a system for amplifying HCV in cultured cells and a system for evaluating the propagation of HCV in cultured cells are necessary, and it is thought that construction of these systems can dramatically advance the above-mentioned research.[0003]HCV is a virus belonging to the flavivirus family of which genome is a single-stranded (+) sense RNA and is known to cause hepatitis C. Recently, it has been revealed that the HCV is classified into many types depending on genotypes or serotypes. Acc...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(United States)
IPC IPC(8): C12N7/08C12N15/86C07K16/10C07K16/08C07K14/05A61K39/12C12N7/04C12N7/00C12Q1/70A61K39/00C07K14/005
CPCC12N7/00A61K39/12C07K14/005C07K16/082C07K16/109C12N15/86C12Q1/707A61K39/00C12N2770/24221C12N2770/24222C12N2770/24231C12N2770/24234C12N2770/24243C12N2770/24251C12Q2600/136C12Q2600/156A61P31/14
Inventor WAKITA, TAKAJIDATE, TOMOKOTANAKA, YASUHITOMIZOKAMI, MASASHI
Owner JAPAN AS REPRESENTED BY DIRECTOR GENERAL OF NAT INST OF INFECT IOUS DISEASES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com